Benzamide, 4-methoxy-N-methyl-3-nitro-

Endpoint summary

Description of key information

Compared to the relative absorbance value of the negative control the mean relative absorbance value was reduced irrelevantly to 93.1% after exposure of the skin tissues to the test item. This value is above the threshold for irritancy of ≤ 50%. Therefore, the test item is not considered to possess an irritant potential.

Relative to the negative control, the test item 4-Methoxy-N-methyl-3-nitrobenzamid did not cause an increase of the corneal opacity and permeability. The calculated mean IVIS was 0.12 (threshold for No Category: IVIS ≤ 3). According to OECD 437 the test item is identified as not requiring classification for eye irritation or serious eye damage i.e. No Category (UN GHS).

Key value for chemical safety assessment

Skin irritation / corrosion

Link to relevant study records

Reference

Endpoint:

skin irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2016

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 439 (In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method)

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: UN GHS (2003, last rev. 2015)

Principles of method if other than guideline:

Based on a “Statement on the Scientific Validity of In Vitro Tests for Skin Irritation” of the European Commission (November 2008), official acceptance of the test method in the EU was achieved and implemented in EU, 2008a, Council Regulation (EC) No 440/2008 of 30 May 2008 laying down test methods pursuant to EC Regulation No 1907/2006 of the European Parliament and of the Council on REACH; 1st ATP 2009: EC Regulation No 761/2009 of 23 July 2009 amending, for the purpose of its ATP, EC Regulation No 440/2008 laying down test methods pursuant to EC Regulation No 1907/2006 of the European Parliament and of the Council on REACH, section B46.

GLP compliance:

yes (incl. QA statement)

Test system:

human skin model

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

25 mg (~ 39 mg/cm2 according to guideline)

Duration of treatment / exposure:

60 minutes.

Number of replicates:

3

Species:

other: reconstituted human epidermis model

Type of coverage:

other: Topical

Preparation of test site:

other: Not applicable

Vehicle:

other: No vehicle used

Controls:

yes

yes, concurrent positive control

yes, concurrent negative control

Amount / concentration applied:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit):
Each approximately 25 mg (~ 39 mg/cm2 according to guideline) of the test item were applied to each tissue, wetted with 25 µL DPBS, and spread to match the surface of the tissue for a complete treatment time of 60 minutes.


NEGATIVE CONTROL
- Amount(s) applied (volume or weight): 30 µL DPBS (MatTek) were used as negative control per tissue.


POSITIVE CONTROL
- Amount(s) applied (volume or weight): 30 µL of a 5% SLS solution in deionised water (MatTek) were used as positive control per tissue.

Duration of treatment / exposure:

60 minutes

Observation period:

Not applicable

Number of animals:

Not applicable

Irritation / corrosion parameter:

other: % relative absorbance

Run / experiment:

1

Value:

82.2

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Irritation / corrosion parameter:

other: % relative absorbance

Run / experiment:

2

Value:

95

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Irritation / corrosion parameter:

other: % relative absorbance

Run / experiment:

3

Value:

102.1

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Results after treatment with 4-Methoxy-N-methyl-3-nitrobenzamid and the controls (exposure interval of 60 minutes):

Dose	Tissue	Absorbance	Absorbance	Absorbance	Mean	Mean
Group	No.	570 nm	570 nm	570 nm	Absorbance	Absorbance
		Well 1	Well 2	Well 3	of	of three wells
					3 Wells	blank
	-	-	-	-	-	corrected
Blank	-	0.037	0.038	0.038	0.037	0.000
Negative Control	1	1.873	1.874	1.815	1.854	1.817
Negative Control	2	2.080	2.059	2.045	2.062	2.024
Negative Control	3	1.924	1.847	1.775	1.849	1.811
Positive Control	1	0.100	0.099	0.099	0.099	0.062
Positive Control	2	0.099	0.108	0.109	0.105	0.068
Positive Control	3	0.108	0.110	0.107	0.108	0.071
Test Item	1	1.535	1.601	1.620	1.585	1.535
Test Item	2	1.761	1.856	1.866	1.828	1.761
Test Item	3	1.881	2.001	2.003	1.962	1.881

Dose	Tissue	Mean Absorbance of 3 tissues	Relative	Relative	Mean
Group	No.	after blank	Absorbance [%]	Standard	Relative Absorbance
		correction*	Tissue 1, 2, 3 ***	Deviation	[% of Negative
				[%]	Control] ***
Negative Control	1		96.4
Negative Control	2	1.884	107.4	6.4	100.0
Negative Control	3		96.1
Positive Control	1		3.3
Positive Control	2	0.067	3.6	6.8	3.5
Positive Control	3		3.8
Test Item	1		82.2
Test Item	2	1.754	95.0	10.9	93.1
Test Item	3		102.1

*         Mean of three replicate wells after blank correction

**       relative absorbance per tissue [rounded values]

***     relative absorbance per treatment group [rounded values]

Interpretation of results:

other: not irritant

Conclusions:

In conclusion, it can be stated that in this study and under the experimental conditions reported, 4-Methoxy-N-methyl-3-nitrobenzamid is not irritant to skin according to UN GHS and EU CLP regulation.

Executive summary:

This in vitro study was performed to assess the irritation potential of 4-Methoxy-N-methyl-3-nitrobenzamid by means of the Human Skin Model Test.

The test item passed the MTT- and the Colour Interference pre-tests.

Approximately 25 mg of the test item were applied to each tissue, wetted with 25 µL of DPBS, and spread to match the surface of triplicate tissue.

30 µL of either the negative control (DPBS) or the positive control (5% SLS) were applied to triplicate tissue each.

The test item and the positive and negative controls were washed off the skin tissues after 60 minutes treatment. After further incubation for about 43 hours the tissues were treated with the MTT solution for 3 hours following about 3 hours extraction of the colorant from the cells. The amount of extracted colorant was determined photometrically at 570 nm.

After treatment with the negative control the absorbance values were well within the required acceptability criterion of mean OD ≥ 0.8 and ≤ 2.8 for the 60 minutes treatment interval thus showing the quality of the tissues.

Treatment with the positive control induced a decrease in the relative absorbance as compared to the negative control to 3.5% thus ensuring the validity of the test system.

The relative standard deviations between the % variability values of the test item, the positive and negative controls in the main test were below 11% (threshold of the "OECD Guideline for the Testing of Chemicals 439:In vitroSkin Irritation: Reconstructed Human Epidermis Test Method”: < 18%), thus ensuring the validity of the study.

Compared to the relative absorbance value of the negative control the mean relative absorbance value was reduced irrelevantly to 93.1% after exposure of the skin tissues to the test item. This value is above the threshold for irritancy of ≤ 50%. Therefore, the test item is not considered to possess an irritant potential.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not irritating)

Eye irritation

Link to relevant study records

Reference

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2016

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 437 (Bovine Corneal Opacity and Permeability Test Method for Identifying i) Chemicals Inducing Serious Eye Damage and ii) Chemicals Not Requiring Classification for Eye Irritation or Serious Eye Damage)

Deviations:

no

Principles of method if other than guideline:

Bovine Corneal Opacity and Permeability (BCOP) Assay, SOP of Microbiological Associates Ltd., UK, Procedure Details, April 1997.
EC Commission Directive 2004/10/EC

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

IIdentification: 4-Methoxy-N-methyl-3-nitrobenzamid
Other Name: NASMA
CAS No.: 333350-60-0
Batch: Op 2/16
Purity: 99.5% (w/w), dose calculation was not adjusted to purity
Appearance: Pale yellow powder
Expiry Date: 14.07.2018 (Statement of Sponsor)
Storage Conditions: At room temperature
Certificate of Analysis: AZ 1027/Toxd1, dated 14 July 2016
Stability in Solvent: Not indicated by the Sponsor

Species:

other: Freshly isolated bovine cornea (at least 9 month old donor cattle)

Strain:

not specified

Details on test animals or tissues and environmental conditions:

Source: Schlachthof Aschaffenburg, 63739 Aschaffenburg, Germany

Controls:

yes, concurrent positive control

yes, concurrent negative control

Amount / concentration applied:

Amount(s) applied (volume or weight with unit): 0.75 mL

Duration of treatment / exposure:

240 minutes

Number of animals or in vitro replicates:

3 corneae per group (test item, negative control, positive control)

Details on study design:

Three corneas were exposed to each 0.75 mL of the test item, the negative, and the positive control for 10 minutes.

SCORING SYSTEM:
Opacity Measurement:
The opacitometer determines changes in the light transmission passing through the corneae, and displays a numerical opacity value. This value was recorded in a table. The opacitometer OP_KiT opacitometer (Electro Design, 63-Riom France) was calibrated as described in the manual and the opacity of each of the corneae was determined by reading each holder placed in the photoreceptor compartment for treated cornea.
After exposure of the corneae to the test groups, after rinsing and further incubation of the corneae for two hours, the opacity value was determined again (t130).

Permeability Determination:
Following the opacity readings, the permeability endpoint was measured as an indication of the integrity of the epithelial cell sheets. After the final opacity measurement was performed, the complete medium was removed from the anterior compartment and replaced by 1 mL of a 0.4% (w/v) sodium fluorescein solution in HBSS. Corneae were incubated again in a horizontal position for 90 minutes in a water-bath at 32 ± 1 °C. Complete medium from the posterior compartment was removed, well mixed and transferred into a 96 well plate and the optical density at 490 nm (OD490) was determined with a spectrophotometer.
The optical density was measured with a microplate reader (Versamax® Molecular Devices) at 490 nm (OD490). The absorbance values were determined using the software SoftMax Pro Enterprise (version 4.7.1).

Data Evaluation

Opacity:
The change of opacity value of each treated cornea or positive and negative control corneae is calculated by subtracting the initial basal opacity from the post treatment opacity reading (t130 – t0).
The average change in opacity of the negative control corneae is calculated and this value is subtracted from the change in opacity of each treated cornea or positive control to obtain a corrected opacity.

Permeability:
The corrected OD490 value of each cornea treated with positive control and test item is calculated by subtracting the average negative control cornea value from the original permeability value for each cornea.


IVIS Calculation:

The following formula is used to determine the IVIS of the negative control:
IVIS = opacity value + (15 x OD490 value)
The following formula is used to determine the IVIS of the positive control and the test item:
IVIS = (opacity value – opacity value mean negative control) + (15 x corrected OD490 value)
The mean IVIS value of each treated group is calculated from the IVIS values.
Depending on the IVIS score obtained, the test item is classified into the following category according to OECD guideline 437:

IVIS UN GHS
≤ 3 No Category
> 3; ≤ 55 No prediction can be made
> 55 Category 1

Criteria for Determination of a Valid Test:

The test will be acceptable if
• the positive control gives an IVIS that falls within two standard deviations of the current historical mean (updated every three months), and if
• the negative control responses result in opacity and permeability values that are less than the established upper limits for background opacity and permeability values for bovine corneae treated with the respective negative control.

Irritation parameter:

in vitro irritation score

Run / experiment:

1

Value:

-0.05

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Irritation parameter:

in vitro irritation score

Run / experiment:

2

Value:

0.04

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Irritation parameter:

in vitro irritation score

Run / experiment:

3

Value:

0.36

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Irritant / corrosive response data:

Relative to the negative control, the test item 4-Methoxy-N-methyl-3-nitrobenzamid did not cause an increase of the corneal opacity and permeability. The calculated mean IVIS was 0.12 (threshold for No Category: IVIS ≤ 3). According to OECD 437 the test item is identified as not requiring classification for eye irritation or serious eye damage i.e. No Category (UN GHS).

Other effects:

None

Results after 240 Minutes Incubation Time

Test Group	Opacity value = Difference (t240-t0) of Opacity		Permeability at 490 nm (OD490		IVIS	Mean IVIS	Proposed UN GHS Category
		Mean		Mean
Negative Control	0		0.055		0.83
	0	0.00	0.052	0.054	0.78	0.82	No Category
	0		0.056		0.84
Positive Control	118.00*		0.323*		122.84
	100.00*		0.110*		101.65	107.73	Category 1
	98.00*		0.047*		98.70
4-Methoxy-N-methyl-3-nitrobenz-amid	0.00*		-0.003*		-0.05
	0.00*		0.003*		0.04	0.12	No Category
	0.00*		0.024*		0.36

 *corrected values

 

*corrected values

Interpretation of results:

other: According to OECD 437 no prediction can be made whether the test item induces serious eye damage (UN GHS: Category 1) or not (UN GHS: no Category).

Conclusions:

In conclusion, according to the current study and under the experimental conditions reported, 4-Methoxy-N-methyl-3-nitrobenzamid is identified as not inducing eye irritation or serious eye damage.

Executive summary:

This in vitro study was performed to assess the corneal damage potential of 4-Methoxy-N-methyl-3-nitrobenzamidby means of the BCOP assay using fresh bovine corneae.

After a first opacity measurement of the fresh bovine corneae (t₀), the 20% (w/v)suspensionin saline of the test item 4-Methoxy-N-methyl-3-nitrobenzamid, the positive, and the negative controls were applied to corneae and incubated for 240 minutes at 32± 1 °C. After the incubation phase the test item, the positive, and the negative controls were each rinsed from the corneae andopacity was measured again (t₂₄₀).

After the opacity measurements permeability of the corneae was determined by measuring spectrophotometrically the transfer of sodium fluorescein after incubation in a horizontal position for 90 minutes at 32 ± 1 °C.

With the negative control (saline) neither an increase of opacity nor permeability of the corneae could be observed (mean IVIS = 0.82).

The positive control (10% (w/v) Benzalkonium chloride in saline) showed clear opacity and distinctive permeability of the corneae (mean IVIS =107.73)corresponding to a classification as “Causes serious eye damage” (CLP/EPA/GHS (Cat 1)).

Relative to the negative control, the test item 4-Methoxy-N-methyl-3-nitrobenzamid did not cause an increase of the corneal opacity and permeability. The calculated mean IVIS was 0.12 (threshold for No Category: IVIS ≤ 3). According to OECD 437 the test item is identified as not requiring classification for eye irritation or serious eye damage i.e. No Category (UN GHS).

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not irritating)

Respiratory irritation

Endpoint conclusion

Endpoint conclusion:

no study available

Justification for classification or non-classification

No Classification

No irritant effects were recorded in a human skin model as well as in a bovine cornal apocity test (BCOP)